The present invention relates to a novel family of immediate early response genes, the use of members of the family in diagnostic and therapeutic applications, in addition to drug design and vaccination protocols.
Normal growth and differentiation of all organisms is dependent on cells responding correctly to a variety of internal and external signals. Many of these signals produce their effects by ultimately changing the transcription of specific genes. One of the major goals of developmental biologists is to define the interactions of gene products and the role they play in regulating cellular differentiation in time and space. Moreover, it is clear that inappropriate expression of many genes that control differentiation during embryonic development can lead to oncogenic transformation. Such genes include members of the growth factor families and components of their signal transduction pathways.
Polypeptide growth factors are members of a growing family of regulatory molecules that have been conserved throughout evolution and are known to have pleiotropic effects which range from stimulation of cell proliferation to control of cell differentiation. Growth factors have been linked to oncogenesis as many of the known oncogenes have been identified as overexpressed and/or mutated forms of growth factors, growth factor receptors or components of their intracellular signal transduction pathways. Oncogenes are thought to be altered such that their product escapes the normal control mechanism(s), resulting in the signalling pathways being permanently switched on. The overall result is uncontrolled cell growth.
The family of fibroblast growth factors (FGFs) consists of a number of members related by sequence and their ability to bind heparin (1). FGFs are involved in a number of cellular activities, including mitogenesis, cell differentiation and angiogenesis (reviewed in 2). In addition, overexpression of FGF in various cell lines leads to phenotypic transformation (3-5).
For example, fgf-3 was identified by its proximity to a preferred integration site of the proviral DNA of the murine mammary tumour virus (MMTV) in MMTV induced mammary carcinomas (Moore, et al., 1986), while fgf-4 was isolated from Kaposi""s sarcoma by its ability to transform NIH 3T3 cells (Delli-Bovi and Basilico, 1987). Some members of the family were identified by their mitogenic activity such as fgf-2, which can cause phenotypic transformation when overexpressed in cultured cells (Sasada, et al., 1988; Neufeld, et al., 1988), thus classifying them as potential oncogenes.
Most of the studies to date have focused on FGF""s mitogenic and transforming activities, however, FGF has also been shown to act as a differentiation factor for embryonic cells (Slack et al., 1987). For example, FGFs have been shown to induce mesoderm differentiation in Xenopus embryonic tissue (6) and many of the initial events in the cellular response during induction are similar to those previously characterized for the FGF-mediated mitogenic response. During mesoderm induction, FGF binds to high affinity cell surface receptors (7) which in turn become phosphorylated on tyrosine (8). The phosphorylated FGF receptor (FGFR) forms a signalling complex by binding a number of intracellular substrates (9) which results in activation of several well-characterized signalling pathways. For instance, protein kinase C becomes activated during FGF-induced mesoderm differentiation (8) as does MAPK (10).
Previously, growth and differentiation had been thought to be mutually exclusive, i.e. when a cell begins to differentiate, it stops dividing. Thus, the elucidation of the mechanisms that regulate the differentiation process may provide may provide valuable information about the molecular signals that are important for arresting cell growth. Further research in this field will contribute to an understanding of how growth factors, such as FGF function during early embryonic development to regulate patterning of mesodermal tissues and highlight differences in the cellular response during growth, differentiation and oncogenesis. It is therefore hoped that by elucidating the molecular mechanisms by which genes regulate developmental processes during embryogenesis, it may be possible to define how misregulation of these genes can lead to cancer.
Recent research has focused on finding means for triggering the immune system to attack cancerous cells, a tactic termed immunotherapy or vaccine therapy. Because immunity is a systemic reaction, it holds the potential to eliminate all cancer cells in a patient""s body, even when they migrate away from the original tumor site or reappear after years of clinical remission. One challenge is that the immune system does not always recognize cancer cells and single them out for attack. A possible solution is to tag cancer cells with certain genes rendering them more visible to the immune system, which can then destroy them.
The immune response involves many different cells and chemicals that work together to destroy in several ways invading microbes or damaged cells. In general, abnormal cells sport surface proteins, called antigens, that differ from those found on healthy cells. When the immune system is activated, B lymphocytes produce antibodies which circulate through the body and bind to foreign antigens, thereby marking the antigen bearers for destruction by other components of the immune system. Other cells, T lymphocytes, recognize foreign antigens as well; they destroy cells displaying specific antigens of stimulate other killer T cells to do so. B and T cells communicate with one another by way of secreted proteins, cytokines. Other accessory cells, antigen-presenting cells and dendritic cells, further help T and B lymphocytes detect and respond to antigens on cancerous or infected cells.
One theory of a means of identifying cancer cells entails the abnormal expression of genes that are normally expressed only very early in development, such as during embryogenesis. If these types of genes are not expressed in normal, healthy adult cells, but are during cancerous growth, then proteins could be expressed that could function as an antigenic marker for immune attack.
Immunizing an organism with DNA coding for this antigen, could train or sensitize the immune system to attack cells expressing these antigens that are only expressed in during cancerous growth. Moreover, sensitive diagnostic means using either labelled polynucleotide probes or antibodies could be developed to detect the polynucleic acid messengers, such as mRNA, indicating the expression of these genes, hence the transformation into cancerous growth.
The subject invention concerns M-MIER gene family and its polynucleotide sequences which encode proteins; members of this gene family are activated in response to fibroblast growth factor (FGF) in an immediate early sequence. As an exemplary member of the M-MIER gene family, er1 is an early response gene that encodes a transcription factor found in the cell nucleus and is activated in response to FGF.
Embodiments of this invention pertaining to the M-MIER gene family comprise:
1) genomic sequences, gene sequences and partial sequences of the members of the mammalian M-MIER gene family;
2) isolated, synthetic M-MIER gene sequences;
3) polynucleotide sequence probes for diagnostic use;
4) polynucleotide sequences for antisense gene therapy;
5) polynucleotide sequences for DNA vaccines;
6) polynucleotide sequences for gene replacement therapy;
7) cloning vectors comprising mammalian M-MIER gene sequences;
8) antibodies to partial mammalian M-MIER gene sequences;
9) antibodies to peptides encoded by M-MIER gene sequences;
10) diagnostic kits comprising nucleic acid probes; and
11) diagnostic kits comprising antibodies to M-MIER proteins.
An object of the present invention is to provide a family of mammalian genes that are transcribed in the immediate early phase of mesoderm induction following exposure to FGF. In accordance with an aspect of the present invention there are provided cDNAs encoding members of this M-MIER gene family.
In accordance with another aspect of the invention there is provided a probe to identify and isolate similar gene sequences.
In accordance with yet a further aspect of the invention there is provided antisense nucleotides to block expression of gene products. In particular, the present invention provides synthetic oligonucleotides, designed to bind to the ER1 consensus DNA binding sequence, GTTTC/GG, that can be used to bind to mammalian DNA to inhibit er1.
In one embodiment of the subject invention, the proteins encoded by the genes described herein can be used to raise antibodies which in turn can be used in diagnostic or therapeutic applications.
In one aspect, the present invention provides a member of the M-MIER gene family: an isolated and purified ER1 polypeptide. Preferably, the polypeptide is a recombinant polypeptide, and more preferably comprises the amino acid sequence of FIG. 22.
In another aspect, the present invention provides an isolated and purified polynucleotide that encodes a M-MIER polypeptide. Preferably, the polynucleotide is a DNA molecule, such as an isolated and purified polynucleotide comprising the nucleotide base sequence for one member of the M-MIER family, ER1, shown in FIG. 22.
The present invention also contemplates an expression vector comprising a polynucleotide that encodes a M-MIER polypeptide. In a preferred embodiment, the polynucleotide is operatively linked to an enhancer-promoter.
Also contemplated is a recombinant cell transfected with a polynucleotide that encodes a M-MIER polypeptide. Preferably, the polynucleotide is under the transcriptional control of regulatory signals functional in the recombinant cell, and the regulatory signals appropriately control expression of the receptor polypeptide in a manner to enable all necessary transcriptional and post-transcriptional modification.
In yet another aspect, the present invention contemplates a process of preparing a M-MIER polypeptide, by producing a transformed recombinant cell, and maintaining the transformed recombinant cell under biological conditions suitable for the expression of the polypeptide.
The present invention also contemplates an antibody immunoreactive with a M-MIER polynucleotide and/or polypeptide. The antibody may be either monoclonal or polyclonal. Preferably, the antibody is a monoclonal antibody produced by recovering the polynucleotide and/or polypeptide from a cell host, expressing the polypeptides and then preparing antibody to the polypeptide in a suitable animal host.
In still another aspect, the present invention provides a process of detecting a M-MIER polynucleotide and/or polypeptide, which process comprises immunoreacting the polynucleotide and/or polypeptide with an antibody of the present invention and a diagnostic assay kit for detecting the presence of a M-MIER polynucleotide and/or polypeptide in a biological sample, the kit comprising a first container means comprising a first antibody that immunoreacts with the M-MIER polynucleotide and/or polypeptide. The first antibody is present in an amount sufficient to perform at least one assay.
Still further, the present invention provides a process of detecting a DNA molecule or RNA transcript that encodes a M-MIER polypeptide by hybridizing the DNA or RNA transcript with a polynucleotide that encodes the polypeptide to form a duplex, and then detecting the duplex.
Still further, the present invention provides a process of screening a substance for its ability to interact with members of the M-MIER family of proteins.
It is a further object of the present invention to provide a diagnostic marker for rapidly proliferating cells. A further aspect of the invention is concerned with a diagnostic kit containing antibodies to the nucleic acid of the invention. Yet a further aspect of the invention is concerned with a diagnostic kit containing antibodies to the protein encoded by the nucleic acid of the instant invention.
In still a further object of the present invention, a DNA binding domain of the protein product of M-MIER family of genes is provided, such as the SANT domain.